The proposed study will focus on matrix vesicles (MVs) which play a role in the mineralization of teeth. Matrix vesicles are submicroscopic, extracellular, membrane invested particles which serve as the initial loci of mineralization of dentin, and of other oral tissues including cartilage and bone. Our lab was involved in the original identification of MVs and in their subsequent isolation and partial characterization. We have provided evidence that matrix vesicle phosphatases (including ATPase, pyrophosphatase and alkaline phosphates) are involved in mineralization. Furthermore, these phosphatases are associated with the vesicle membrane and can be solubilized with preservation of enzymatic activity and reconstituted into vesicles with restitution of calcium-depositing activity. ln work supported by the present grant we have: 1) Developed an in vitro method to study isolated MV calcification under conditions which allow hydroxyapatIte (HA) mineral deposition; 2) Developed a monoclonal antibody against MV alkaline phosphatase (ALPase), and used it to purify ALPase to chemical homogeneity in quantities never previously available; 3) Used the monoclonal antibody in a first attempt to iummunolocalize ALPase at EM levels in membranes of cells and MVs; and 4) Devised a mammalian cell culture system to follow MV biogenesis and release (presumably from plasma membrane) and MV calcification in vitro. Proposed new studies include: 1) An attempt to determine the location and orientation of ALPase within the MV membrane by immunocytochemistry using new antibodies, and biochemically by application of specific enzymes, extractants and detergents which indicate whether MV ALPase is a transmembrane protein; 2) An attempt to reconstitute MV ALPase into proteoliposomes with restoration of enzyme activity and calcifiability; and 3) The use of mammalian chondrocyte cultures of examine MV biogenesis in greater detail, including possible metabolic controls. This is a fundamental study of the mechanism by which dentinal and skeletal forms of mineralization are brought about. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific disease states which abnormal calcification occurs.